dietary docosahexaenoic acid is retroconverted in man to eicosapentaenoic acid, which can be quickly...

PROSTAGLANDINS

DIETARY DOCOSAHEXAENOIC ACID IS RETROCONVERTED IN MAN

TO EICOSAPENTAENOIC ACID, WHICH CAN BE QUICKLY TRANS-

FORMED TO PROSTAGLANDIN 13.

Sven Fischer, Axe1 Vischer, Vera Preac-Mursic* and Peter C. Weber

Medizinische Universitatsklinik Innenstadt, 8000 Miinchen 2, Ziemssenstrasse 1, F.R.G.

*Max-von-Pettenkofer-Institut fur Hygiene und Medizinische Mikrobiologie, 8000 Milnchen 2, Pettenkoferstrasse 9 a, F.R.G.

ABSTRACT

In a 24 h kinetic studv docosahexaenoic acid (DCHA, C22:6n-3) or eicosapentaenoic acid (EPA, C20:5n-3) were given in a single dose to healthy male volunteers. PGI

a -M, the main uri-

nary metabolite of prostaglandin I was be1 w the detection limit in the control periods, but aas excreted already in the first 4 h after ingestion of DCHA or EPA and decreased thereafter. Excretion of PGI -M did not change significantly. In a second dietary trial i?iCHA and EPA were given cross-over to 7 healthy male volunteers for 6 days. PG13-M was formed after DCHA and EPA in amounts of 35 and 20 % of PGI,-M and showed a considerable interindividual variation. TheLstructure of PGI -M was verified by independant biochemical synthesis. Our dat$ indicate that dietary DCHA is retroconverted to EPA in man, which is quickly transformed - like dietary EPA itself - to prostaglandin 13. DCHA may therefore serve as a precursor fatty acid for EPA and its cyclooxygenated and lipoxygenated products.

INTRODUCTION

EPA and DCHA are the main n-3 polyunsaturated fatty acids in triglycerides of maritime origin. After dietary enrichment in man EPA like arachidonic acid may play a more functional role and can be metabolised to eicosanoids by endothelium, thrombocytes and neutrophils (1,2 ). However, DCHA seems to be of more structural importance and is liberated from platelets only in traces upon stimulation with thrombin (3). In the following 24 h kinetic trial and in a one week intervention study (4) we compared formation of prostacyclin of the two and three series after dietary DCHA and EPA. We found after both dietary DCHA and EPA a rapid formation of prostaglandin 13.

SEPTEMBER 1987 VOL. 34 NO. 3 367

PROSTAGLANDINS

STUDY PROTOCOLS, MATERIALS AND METHODS

STUDY PROTOCOLS

a) 24 h kinetic study DCHA-ethylester were given orally in a single dose to 4

heilthy male volunteers Urine samples were collected before, 4, 8, 24 and 26 h after'supplementation and PGI

212 -M in urine

was assayed. Also 6 g EPA-ethylester were given a single dose to 4 healthy male volunteers. Urinary PGI before,

-M was assayed 4. 8, 24 and 26 h after ingestion of EPA?

b) 6 day study In a double brind, randomized study 7 healthy male volunteers ingested 6 g DCHA- or 6 g EPA-ethylester daily for 6 days with a 10 week washout-period. They supplemented their diet with 100 mg vitamin E daily, but refrained from all other drugs and excluded dietary fish one week prior and during the study periods. Urinary analyses were performed before and 24 h after ingestion of the capsules for 6 days and were done with- out knowledge of the type of dietary supplementation (4). PG12,3-M in urine was assayed by combined GC-MS.

MATERIALS

Soft gelatine capsules of EPA-ethylester (0.5 g. 83 % pure; C22:6n-3, 4.2 X; C22:5n-3, 0.5 X; C16:0.8 %) and DCHA-ethylester (0.25 g, 90 % pure; C22:5n-3, 5.5 %; EPACl X; C20:4n-6, ~1 %; C22:5n-6-t X) were kindly provided by R/P Scherer, Eberbach, F.R.G. and by Nippon Oil and Fats Co., Tokyo, Japan, respectively. Fatty acid content of capsules was analyzed by Bas-liquid chromatography. 2,3-dinor-6-keto-19,19*, 20,20'- H4-PGF and Mycobakterium rhodochrous were kinqdy provided

by Dr. F?F. Sun, The Upjohn Co.. Kalamazoo, Ml. C-EPA (40-60 mCi/mmol) were from NEN, Dreieich, F.R.G. EPA was from Sigma-Chemie and collagen from Hormon-Chemie, Munich, F.R.G.

METHODS

Mass spectrometric analysis of urinary PGl2,3-M (24 h and 6 day study)

PGI -M (2,3-dinor-6-keto-PGF 6-k&o-PGF oc ).

1 the main uri ary metabo ites of dienoic A”

) and PGl?;M (A17-2,3-dinor-

and trieno c prostacyclin. were analyzed by combined capillary GC-MS in urine collected over 24 h (6 day study) and in fractionated periods over 26 h (24 h kinetic study). 100 ml samples were extr$cted as described (5) after addition of 25 ng 19,19' 20,20'- H -2,3-dinor-6-keto-PGF as internal standard. The methoxim -pentafluorobenzylest$?-trimethyl- ? silylether-derivatives were prepared as described (6) and fragments m/z 590, 586 and 584 (M-181 (C H F )) of deute-

zM2wsre monitored rated internal standard, PGlB;M and PGl3 in the negative ion chemical Ionization mode (isobutane). The Gi-MS-system was a Finnigan MAT 445 (Bremen, F.R.G.) equipped with a SE 30 fused silica capillary column (25 m, 0.25 mm i.d.). Operating conditions of the GC-MS-system were: injection port 290°C. interface 28O'C. ion source 2DD°C, electron impact energy 150 eV, current of emmission 1.2 mA, electron multiplier voltage 2.5 kV.

368 SEPTEMBER 1987 VOL. 34 NO. 3

PROSTAGLANDINS

Biochemical synthesis of authentic PGI3-M

a) Synthesis ofAj;(-6-keto-PGF DA (100 ) d "C-EPA (2 5 ?+-106 . cpm) were incubated with 7 g fresh:; pF:pared sliced calf aorta in 25 ml PM 16 glucose buffer for 4 h at 37'C (7). The supernatant was acidified with HCOOH to pH 3.5 and lipids were extracted by Sep-Pak- reversed phase cartridges and eluted with ethylacetate (8). A17-6-keto-PGFd,, which contained 6-keto-PGF .)"' formed from endogenous arac idonic acid, was further purl led by reversed phase HPLC using the solvent system water/acetonitrile/f6etic acid in the ratio 760/240/2 (9 ). Fractions containing C-n17- 6-keto-PGF,, were collected and Iyophilized.

b) Synthesis ofA17-2,3-dinor-6-keto-PGF (PGI -M) The HPLC-effluent containing about 2 ugB%&FPGF was lyophilized, dissolved in 20 ul ethanol and added ts 1 ml phosphate buffer (50 mM. pH 7.4) containing Mycobakterium rhodochrous, which had been cultured in 10 ml tryptonelyeast- medium for 72 h at 25°C in the dark (13). After 3-4 days at 25°C in the dark the incubation was terminated with 5 ml acetone and lipids were extracted after acidification to pH 3.5 (HCOOH) with ethylacetate. PG13-M contaminated with PG12-M was transformed to the Me, MO, Me rated by gas-liquid chromatograph .?

Si-derivative (1) and sepa- on a Carbowax CP51 column

from PG12 -M as described (1).

RESULTS

24 h KINETIC STUDY -~ E3-M is already formed in the first 4 h after dietary DCHA

Fig. 1 shows the excretion of PGI -M and PGI -M after a single oral dose of 6 g DCHA in 4 volunt ers. 5 PGIK-d,was at levels of 130 rig/g creatinine and did not change sig ificantly in the 24 h after dietary DCHA. PGI -M was below the detection limit in the control period, increas d rapidly to 30 % (mean) of PGI -M in 2 the first 4 h after dosing and declined thereafter. At 26 h PGI -M was again below the detection limit. Formation of PG13-M shoaed marked interindividual variations after dietary DCHA.

E3-M is also formed in the first 4 h after dietary EPA

After a single dose of 6 g EPA a partly different collective of 4 volunteers showed a PGI -M excretion of 80 rig/g creatinine which did not change signifi .? antly during the collection periods. PGI -M was clearly detectable in the first 4 h after ingestion of t PA in 2 volunteers but only one individual showed high formation of 35 % PG13-M as compared to PG12-M. Already 8 h after the single dose of EPA-ethylester PGI -M was below the detection limit, which was about 3 % of PG?2-M.


PROSTAGLANDINS

bg DCHA-ETHYLESTER

SINGLE DOSE

100 , 8

!__---_L_& PG’3-M CONTROL 0 - Lh L-Bh 8 -2Lh ZL-26h

Y

.f

c _ 300 ;; a

;

01 200

?

100

* . . .

. *

CONTROL 0-Lh L-Oh B-2Lh ZL-26h

Fig. 1: Kinetics of the urinary excretion of PGI -M and PGI -M after a single oral dose of 6 g DCHA-ethylester ‘i; n=4 volunz 1 teers. PGI -M was below the detection limit in the control period and a ter 26 h. Mean values are also indicated. 3

6 DAY INTERVENTION STUDY

PGI -M is formed after both dietary EPA and DCHA in the 6 day ma,

Excretion of PGI -M was 69tl9.5 and 68233 rig/g creatinine in the control .$ pet-i ds and did not change significantly after 6 days of dietary EPA or DCHA (Fig. 2). PGI -M, which was be!ow the detection limit in the control pe J. lads, rose to 14-9.7 rig/g creatinine aft$r dietary EPA. After dietary DCHA excretion of PG13-M was 27-24.8 rig/g creatinine (Fig. 2) Values are X*S.D. However there were considerable interindividual variations in the iormation of PG13-M especially after dietary DCHA.


PROSTAGLANDINS

150

100 0, c ._ C ._

i 50

-T 2

0

EPA OCHA 6 grams for 6 days

150

100

50

0

Fig. 2: Excretion of PGI -M (0 and PGI -M (0) in 7 volunteers betore and after suppleme tation i; of the1 '? diet with 6 g EPA- or OCHA-ethylester for 6 days. Control and intervention values are connected by a line. in the control periods. The Aean -5.E.M. are indicated.

PGI -M was below the detection limit

BIOCHEMICAL SYNTHESIS OF AUTHENTIC PGI3-J

Fig. 3 shows the biochemical synthesis of authentic PGI -M The yield of 317-6-keto-PGF by incubation of endogenaus' EPA with bovine aorta was ard$nd 2 % (n=4 experiments) as calculated from the radioactivity recovered after HPLC. The ratio of i.l7-6-keto-PGF Qenous arachldonic acid 4%~

to 6-keto-PGFl, formed from endo- about 1 to 4. After incubation of

Al7-6-keto-PGF with Mycobakterium rhodochrous (n=4 experiments) the yieis of the 0-oxidation to 2,3-dinor- iZl7-6-keto-


PROSTAGLANDINS

PGF (PGI -M) was varying largely between 5 and 30 % PGI -M syntffesizea biochemically showed after separation from PG13-M on a capillary Carbowax CP 51 column as the Me MO, Me Si-* derivative the same GC-properties as PGI -M exiracted from human urine. It furthermore exhibited ths same characteristic muitiple ion selection tracings in the EI-mode $t m/z 584 (M -15), 530 (M -69), 440 (M+-69-90) and 350 (M -69-2 x 90) as endogenous PG13-M isolated from urine.

AORTIC SLICES + C 20 : 5 n- 3

Al7-6-keto-PGF,=

1

Mycobakterium rhodochrous

617-2,3-dinor-6-keto-PGF,a

PG13-M

Fig. 3: Biochemical synthesis of authentic PGI -M for compa- rison with endogenous PG13-M isolated from urin 2 .

DISCUSSION

In a 24 h kinetic study and in a dietary intervention study of 6 davs Durified OCHA- and EPA-ethvlester were aiven to healthy-maie volunteers and endogenous prostacyclin formation of the two and three series was analyzed by measuring the main urinary metabolites PG12-M and PG13-M.

Excretion of PGI -M in the 24 h kinetic study was already the hiahest in t e first 4 h urinarv collection oeriod after il in

Fi estion of a single dose of eithet purified DCHA- or EPA-

et vlester and decreased thereafter. It was below detection limit after 24 h. This may perhaps indicate that normal endogenous synthesis of prostaglandin I berated phospholipid-bound EPA in c ntrast to high levels of 3

is not derived from li-

prostaglandin I formed after stimulation (11). dietary interve$tion study formation of PGI

In the 6 day

.a -M after ingestion

of purified DCHA and not only after ingesti n of EPA also indicated a retroconversion of DCHA to EPA in man. Excretion of


PROSTAGLANDINS

PGI -M did not change significantly as had been observed pre- vio 6 sly after dietary cod liver oil (1). For quantitation of

%c3 -M by combined capillary GC-MS the pentafluorobenzyl-

-derivatives were monitored in the negative ion chemical ionization mode. The special fragmentation of the derivatives yielding the intact prostanoid molecule (6) and the use of deuterated internal standard enabled us to quantify prostacyclin of the two and three series simultaneously.

The retroconversion of DCHA to EPA may take place in a short time in the liver and has so far only been observed in the rat (12) or in vitro by cultured human retinoblastoma cells (13). It is also paralleled by the retroconversion of dietary doco- sapentaenoic acid (C22:jn-6) to arachidonic acid (C20:4n-6) in the rat (14). The retroconversion of DCHA in vivo in man indicates that DCHA may serve as a precursor fatty acid for the formation of EPA and its cyclooxygenated and lipoxygenated products. The mechanism of retroconversion as it,$as been sug- gested from experiments with uniformly labelled C-DCHA in the rat (12) should also apply to man. It would explain why dietary EPA is not elongated and desaturated to DCHA in the free and phospholipid-bound fatty acids in man (4).

The identity of urinary PGI =-M was oroven bv indeoendent biochemical synthesis in vitro! From endogenous EPA and bovine aorta C17-6-keto-PGF was synthesized which was transformed via O-oxidation by My%bakterium rhodochrous (10) to 2,3-dinor- C:17-6-keto-PGF tographic proper

$" This compound showed the same gas chroma- ies as PGI -M extracted from urine and exhi-

bited an identical fragmentition pattern when subjected to EI- mass spectrometry.

In both the 24 h kinetic study and in the 6 day dietary intervention trial the interindividual variability of PGI3-M formation was high. This points to a different resorption of the ethylesters in the gastrointestinal tract and/or different hydrolase-activities in each individual. The ethylesters of EPA and DCHA prepared from fish oils by various purification steps are the first available purified forms of these n-3- fatty acids for nutritional studies. However, ethylesters are rather "unphysiolopic" compounds and the mechanism of their resorption is largely unknown.

ACKNOWLEDGEMENT

We thank I. Kurzmann, U. Katzner and A. Schmidt for expert technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft.


PROSTAGLANDINS

REFERENCES

I. Fischer, S., and P.C. Weber. 1984. Prostaglandin I formed in vivo in man after dietary eicosapentaeno?c'icid. Nature (Lond.), 307: 165-168.

2. Strasser, T., Fischer, S., and P.C. Weber. 1985. Leukotriene B is formed in human neutrophils after dietary supplemen- tjtion with eicosapentaenoic acid. Proc. Natl. Acad. Sci. USA, 82: 1540-1543.

3. Fischer, S., v.Schacky, C., Siess, W., Strasser, T., and P.C. Weber. 1984. Uptake, release and metabolism of docosahexaenoic acid (DHA, C22:6w-3) in human platelets and neutrophils. Biochem. Biophys. Res. Comm. 120: 3, 907-918.

4. v.Schacky, C., and P.C. Weber. 1985. Metabolism and effects on platelet function of the purified eicosapentaenoic and docosahexaenoic acids in humans. J. Clin. Invest. 76: 2446-50.

5. Falardeau, P., Oates, J-A., and A.R. Brash. 1981. Quantita- tive analysis of two dinor urinary metabolites of prostaglandin 12. Anal. Biochem. 115: 359-367.

6. Waddell, K.A., Blair, I.A., and J. Welby. 1983. Combined capillary column gas chromatography negative ion chemical ionization mass spectrometry of prostanoids. Biomed. Mass Spectrom. IO: 2, 83-88.

7. Adv. Prostaglandin Thromboxane Res. 5: 39-94. Measurement of prostaglandins, thromboxanes, prostacyclin and their metabolites by gas-liquid chromatography-mass spectrometry. Ed. Frolich, J.C., Raven Press, New York. 1978.

8. Powell, W.S. 1980. Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecyl-silyl silica. Prostaglandins 20: 947-957.

9. Fischer, S., Scherer, B., and P.C. Weber. 1982. PrOStaCyClin metabolites, in urine of adults and neonates, studied by gas chromatography-mass spectrometry and radioimmunoassay. Biochim. Biophys. Acta, 710: 493-501.

10. Sun, F.F., Taylor, B.M., Lincoln, F.H., and O.K. Sebek. 1980. Preparation of two dinor-PG12 metabolites from 6-keto-PGF,... by Mycobakterium rhodochrous. Prostaglandins 20: 729-736.

11. Crawford, M.A. 1983. Background to essential fatty acids and their prostanoid derivatives. Brit. Med. Bull. 39: 3, 210-213.

12. Schlenk,H., Sand, D.M., and J.L. Gellerman. 1969. Retrocon- version of docosahexaenoic acid in the rat. Biochim. Biophys. Acta 187: 201-207.


PROSTAGLANDINS

13. Yorek, M-A., Bohnker, R.R., Dudley, D.T., and A.A. Spector. 1984. Comparative utilization of n-3 polyunsaturated fatty acids by cultured human y-79 retinoblastoma cells. Biochim. Biophys. Acta 795: 277-285.

14. Kunau, W.H. 1968. Uber die Synthese der an allen Doppel- bindungen tritiummarkierten 4.7.10.13.16-Docosapentaen- slure und ihre Umwandlung in 5.8.11.14-Eicosatetraensaure bei fettfrei ernahrten Ratten. Hoppe-Seyler's Z. Physiol. Chem. 349: 333-338.

Editor: E. Anggard Received: 7-10-87 Accepted: 7-10-87


dietary docosahexaenoic acid is retroconverted in man to eicosapentaenoic acid, which can be quickly...

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